PROJECT SUMMARY Antibodies are critical components of immune defense and are mediators of vaccine-elicited protection as well as autoimmunity. The heavy and light chains that comprise an antibody molecule are encoded by germline immunoglobulin (Ig) gene fragments that recombine and undergo somatic mutation thereby generating billions of specificities able to contend with a multitude of foreign antigens. The majority of the diversity lies within the antigen-binding (Fab) portion of antibodies although variation also exists within the conserved (Fc) region of the heavy chain that interacts with cellular receptors to mediate non-neutralizing functions. Diversity within both regions impacts on antibody function. Given the protective role of antibodies in HIV and other infections, we propose to mine the depth of the antibody repertoire and study how Fc diversity impacts antiviral activity. It is well known that Africans are more genetically diverse than other populations and yet there is a scarcity of data on African genomes including Ig genes. Our preliminary data using samples from an ethnic Zulu population residing in South Africa has revealed that approximately half of the heavy chain variable (IGHV) genes as well as a significant number of single nucleotide polymorphisms (SNPs) in the Fc are not recorded in public databases. Many of these novel IGHV alleles occur at high frequency and in some cases are highly expressed. Importantly, we have shown that they are used to make functional antibodies. We hypothesize that there is significant undiscovered variability within the African Ig gene repertoire some of which will impact on the functionality of anti-HIV antibodies. To explore this, we propose to sequence the entire rearranged, antigen-nave heavy and light variable chain gene repertoires in 40 individuals of Zulu ethnicity using Illumina MiSeq. Data will be analyzed using a new bioinformatics tool called IgDiscover that will enable the identification of novel and known germline variable and joining region gene segments from both heavy and light chains that are expressed by functional antibodies. Variability in the Fc region of IgG and IgA will be analyzed using SNP analysis in 100 Zulu individuals. Furthermore, the Fc regions from individuals with novel IgG3 SNPs will be sequenced and used to make fully native antibodies for functional studies, including antibody-dependent cellular cytotoxicity (ADCC), phagocytosis and neutralization. In this way the diversity within both the Fab and Fc regions will be analyzed and the impact on function assessed. This project will contribute new knowledge on African germline immunoglobulin genes, which is essential for a fully comprehensive immunogenetics database. These studies will be of great benefit to the HIV research community as they embark on major vaccine efficacy trials as well as to others studying the immune responses to infection, vaccination, cancer and auto-immunity in this population.